A flow-cytometry-based analytical instrument comprises a flow cytometer, a blood analyzer, a urine analyzer or a particle analyzer etc, and utilizes a photosensor to collect and analyze two-dimensional or multi-dimensional optical signals from particles so as to identify different particles in the liquid for classification. As shown in FIGS. 1a and 1b, the existent flow-cytometry-based analytical instrument ordinarily comprises an illumination unit 1, a flow chamber 2 and a photodetection unit (not shown), in which the illumination unit 1 generally consists of a light source 11 and a light beam focusing module 13. The illumination unit 1 is arranged to provide an irradiating light beam, which is projected into a through hole 21 in the flow chamber 2 to form an irradiated area for detection after being shaped by the light beam focusing module 13. When cells flow through the irradiated area, the irradiating beam irradiates cells to cause scattering or initiate fluorescence emission etc, wherein the direction in which cells flow is defined as Y direction, that in which the light beam spreads is defined as Z direction, and that simultaneously perpendicular to both directions in which cells flow and the light beam spreads is defined as X direction. The light beam focusing module can focus Gauss light beams from the light source 11 into the irradiated area which is equal in size to that of a cell in Y direction and to that of the inner wall of the through hole 21 in the flow chamber 2 in X direction, wherein light energy is highly concentrated. Thus, when cells pass through this area, a scattered signal and a fluorescence signal in rather great intensity are easily formed for receiving by the photodetection unit.
The flow chamber 2 is provided with a through hole 21 through which cells can flow, and in which the cells are encased into the sheath fluid based on the fluid focusing principle such that the cells can pass through the irradiated area one by one. In the irradiated area, particles will generate different optical signals as irradiated by the laser light, such as a forward scattered signal (FSC), a side scattered signal (SSC) and a multipath fluorescence signal (FL) etc.
The photodetection unit is disposed to collect a variety of optical signals generated in the flow chamber 2 and convert them into electrical signals, and then transmit these electrical signals to subsequent analytical systems for processing and analyzing to obtain parameters of various cells existent in the fluid, thereby processing accounting and classifying, etc.
In the prior art, the methods are all concerned with using two mutually perpendicular cylindrical lenses to focus the emitted light beam into an elliptical spot to irradiate cells. The Gauss light beams converge at the through hole 21 in the flow chamber 2 in X direction through one cylindrical lens and near the flow chamber 2 in Y direction through the other cylindrical lens. As shown in FIG. 2, the spot in the flow chamber is elliptical, and is approximately equal in size to the cell diameter in Y direction and to that of the inner wall of the through hole 21 in the flow chamber 2 in X direction, and the optical field distribution in X and Y directions is substantially the Gauss distribution.
In the prior art, only the size of the irradiated spot are considered in use of a set of cylindrical lenses to focus the Gauss light beams, without taking the distribution of light intensity in different directions into account. As the use of cylindrical lenses cannot realize the “flat-top” distribution of light intensity, the light intensity in X and Y directions is still presented in the Gauss distribution. Thus, when particles of the same kind pass through different positions of this irradiated area, scattering signals or fluorescence signals in different intensity are formed due to different light intensity irradiated thereon, thereby rendering a misjudgment on the particle. Said misjudgment can be explained via FIGS. 3 and 4. FIG. 3 shows the shape of the irradiated spot and the distribution of light intensity in X direction, in which particles A and B of the same kind enter different positions of an irradiated area 3, and light energy irradiated on particle A in the center of the spot is different from that on particle B away from the center of the spot due to non-uniformity of the distribution of light intensity. As shown in FIG. 4, that will render particle A in the center of the spot and particle B away from the center of the spot to respectively form different scattering signals, i.e. respectively form scattering signal A and scattering signal B. Then, subsequent analytical modules are likely to determine particle B different from particle A based on the scattering signals, thereby causing an error.